other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

RANGE FINDING TESTS:- Compound solubility: Test item was used undiluted and freshly prepared as a solution at the concentrations of 25 and 50% in acetone/olive oil 4:1- Irritation: Mouse treated with diluted test item for three consecutive days (Days 1, 2 and 3) showed no signs of systemic toxicity. Very slight erythema on both ears and a greater than 25% increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 50% w/w in acetone/olive oil 4:1. No visual skin irritation or irritation idicated by an equal to or greater than 25% increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 25% w/w in acetone/olive oil 4:1. Based on this information the dose levels selected for the main test were 25%, 2.5% and 0.25% w/w in acetone/olive oil 4:1.

MAIN STUDY- Name of test method: Local Lymph Node Assay- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION: The mice were treated by daily application of 25 µL of test material at concentrations of 0 (vehicle control), 25, 2.5 and 0.25% to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). On Day 6, all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse. Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. Lymph node cells were washed with PBS and precipitated with 5 % (w/v) trichloroacetic acid (TCA) for radioactive material at 4 °C. Pellets were re-suspended in 1 mL TCA and transferred to 10 mL of scintillation fluid (Optiphase ‘Trisafe’). 3HTdR incorporation was measured by β -scintillation counting. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

No data

Results and discussion

Positive control results:

Stimulation index for α-hexylcinnamaldehyde at 25 % v/v in acetone/olive oil 4:1 was 9.96 and classified as a sensitiser.

other: DPM per group for vehicle, 0.25, 2.5 and 25% were 3188.19, 4332.36, 5172.42, and 5886.25, respectively.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under these test conditions,the substance is not classified as sensitizing to the skin according to CLP Regulation (EC) No. 1272/2008

Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear, according to the OECD Guideline 429 and EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay).

Method

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/w, this concentration was selected as the highest dose level to be investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 uL (25 uL per ear)) of the test item as a solution in acetone/olive oil 4:1 at cocnentrations of 0.025, 2.5 and 25% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using α-

hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

Results

No mortality and no clinical signs were observed during the observation period. Body weight of the animals was unaffected by the test item treatment. The stimulation index for 0.25, 2.5 and 25% were 1.36, 1.62 and 1.85, respectively. The positive control (α-hexylcinnamaldehyde, 25%) exhibited evidence of sensitisation (SI = 9.96) indicating the validity of the study. In this study, the substance is not a skin sensitizer in mice.

Conclusion

The test item was considered to be a non-sensitizer under the conditions of the test.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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